The insulin receptor is encoded by a single copy gene located on chromosome 19 in the human. In addition, at least two other highly homologous genes have been identified. One gene encodes the type 1 insulin-like growth factor (IGF) receptor. The third gene in the family encodes an orphan receptor tyrosine kinase that has been named the insulin receptor-related receptor (IRR). We have cloned the cDNA molecules encoding human and mouse IRR. Generally, the predicted structure of IRR is similar to those of the receptors for insulin and IGF-1. When cells expressing recombinant IRR are incubated with vanadate (an inhibitor of phosphotyrosine phosphatases), this leads to an increase in the phosphotyrosine content of IRR. However, the phosphotyrosine content of IRR. However, the phosphotyrosine content of IRR is not increased by incubating the cells with insulin, IGF-1, or proinsulin. Nevertheless, because IRR may be co-expressed in the same cells as the homologous receptors for insulin and insulin-growth factor-I (IGF-1), we inquired whether IR might be activated via formation of hybrids with insulin receptors. To test this hypothesis, we co-expressed the human IRR and the human insulin receptor (IR) in NIH-3T3 cells. Although IRR/IR hybrid receptors were detected in these cells by using immunoprecipitation techniques, only a small proportion of each receptor was assembled into hybrids. While insulin was capable of stimulating insulin receptor autophosphorylation in these cells, there was no detectable increase in the total phosphotyrosine content of IRR. We conclude that the IRR/IR hybrid receptor does not play a major role in IRR signal transduction in response to insulin in NIH-3T3-hIRR/hIR cells. These observations are consistent with the hypothesis that there is a physiological ligand that binds to the extracellular domain of IRR, thereby specifically its tyrosine kinase activity. To pursue this hypothesis, we used an assay based upon the ability to stimulate autophosphorylation of IRR, and have accomplished about 100-fold purification of a protein factor from rat brain that is a candidate to be the ligand for the IRR.